The invention relates to specific forms of a serum and glucocorticoid-inducible serine/threonine protein kinase called Sgk.
The ability of mammalian cells to sense and respond to dynamic changes in hormonal and nonhormonal environmental stimuli requires individual regulatory molecules that can coordinately integrate the signals emanating from diverse cellular pathways. The availability and function of these signal transduction components ensures the biological specificity and flexibility that allows cells to activate physiologically appropriate responses. We have established that one such critical intracellular signaling component is the serum and glucocorticoid-inducible serine/threonine protein kinase, Sgk, which we isolated from mammary tumor cells as a novel transcriptionally regulated protein kinase (Webster, et al. 1993a. J Biol Chem 268:11482-5, Maiyar, A. C., et al. 1996. J Biol Chem 271:12414-22, Maiyar, A. C., et al. 1997. Mol Endocrinol 11:312-29).
We disclose herein that diverse steroid hormone and phosphosphorylation-dephosphorylation cascades that can evoke anti-proliferative, proliferative and/or environmental stress responses converge on Sgk to selectively regulate its transcription, enzymatic activity and subcellular localization in a stimulus-dependent manner. In normal and transformed cells, glucocorticoids, serum, hyperosmotic stress and UV radiation stimulate sgk promoter activity through specific regulated elements. As part of the glucocorticoid growth arrest response, an inactive hypophosphorylated form of Sgk is produced that is exclusively localized to the cytoplasmic compartment. In serum/growth factor-treated cells, Sgk is selectively phosphorylated and enzymatically activated as a down stream component of the phosphoinositide 3 kinase (PI 3-kinase) signaling cascade, which places Sgk in both a proliferative and a critical cell survival pathway. Hyperosmotic stress induces an enzymatically active Sgk that resides in the cytoplasm, whereas, in serum treated cells the active Sgk shuttles between the nucleus and the cytoplasm in synchrony with the cell cycle. Sgk functions as a critical point of cross talk between cellular cascades both by catalyzing the phosphorylation of specific substrates and by selectively interacting with distinct sets of nonsubstrate targets in a stimulus and cell compartment-specific manner. The invention relates to methods and compositions related to these novel Sgk functionalities.
The invention provides methods and compositions relating to activated Sgk and the association of activated Sgk activity with proliferating cells. In one embodiment, the invention provides methods for inhibiting the growth of an undesirably proliferating cell comprising an amount of activated Sgk, said method comprising the step of contacting the cell with a specific inhibitor of activated Sgk whereby the amount of activated Sgk activity in the cell is reduced. A wide variety of specific inhibitors is disclosed, including dominant negative mutants of activated Sgk, an Sgk mutant or fragment thereof comprising an unphosphorylatable residue corresponding to Thr256 of native Sgk, and an active Sgk-specific antibody or antibody fragment, especially intrabody, etc. A wide variety of methods may be used to contact the target cell with the inhibitor, especially introducing into the cell a polynucleotide encoding the inhibitor under conditions whereby the inhibitor is expressed in the cell.
In another embodiment, the invention provides methods for classifying a proliferating cell as subject to excess activated Sgk activity comprising the step of detecting an above-normal amount of activated Sgk activity in the cell. A wide variety of methods may be used to specifically detect the activated Sgk activity, especially the use of activated Sgk-specific antibodies. In a particular embodiment, the classification and inhibition methods are combined.
In yet another embodiment, the invention provides methods and mixtures for modulating the interactions of Sgk and Sgk-interacting proteins (SIPs), such as Pendulin, PS2 and IF-1 proteins.
The following descriptions of particular embodiments and examples are offered by way of illustration and not by way of limitation. Unless contraindicated or noted otherwise, in these descriptions and throughout this specification, the terms xe2x80x9caxe2x80x9d and xe2x80x9canxe2x80x9d mean one or more, the term xe2x80x9corxe2x80x9d means and/or.
The general methods for inhibiting the growth of an undesirably proliferating cell involve contacting the cell with a specific inhibitor of activated Sgk whereby the amount of activated Sgk activity in the cell is reduced. A wide variety of cell types in in vitro and in situ contexts may be targeted; the suitability of a given cell type and context is readily determined empirically as described and exemplified herein. Particularly suitable cellular targets are shown to comprise elevated levels of activated Sgk and include proliferating reproductive tumor cells, such as breast and prostate cancer cells and lung, kidney, intestine and colon cancer cells.
A wide variety of sgk inhibitors may be used in the methods, so long as the inhibitor is effective, i.e. the effective amount of activated sgk activity in the cell is reduced, and compatable with the target context, e.g. having tolerable toxicity to the host. A reduction in activated sgk activity may be verified by any convenient methods, such as the use of activated Sgk specific antibodies in western blots. Activated Sgk and hyperphosphorylated Sgk both refer to enzymatically active Sgk, empirically defined as the ability to phosphorylate a substrate (Sgktide, below). Activated Sgk is shown to require phosphorylation at the residue corresponding to Thr256 in the rat kinase. Note that for clarity, all residues numerically referenced herein are of rat Sgkxe2x80x94the corresponding residues of alternative species, e.g. human, are readily noted from alignments.
The subject inhibitors comprise a subset of binding agents specific to the subject kinase proteins including substrates, agonists, antagonists, natural intracellular binding targets, etc. Such Sgk-specific binding agents are useful in a variety of diagnostic and therapeutic applications, especially where disease or disease prognosis is associated with improper utilization of a pathway involving Sgk. Novel Sgk-specific binding agents include Sgk-specific receptors, such as somatically recombined polypeptide receptors like specific antibodies or T-cell antigen receptors (see, e.g Harlow and Lane (1988) Antibodies, A Laboratory Manual, Cold Spring Harbor Laboratory) and other natural intracellular binding agents identified with assays such as one-, two- and three-hybrid screens, non-natural intracellular binding agents identified in screens of chemical libraries such as described below, etc. Agents of particular interest modulate Sgk function. Exemplary Sgk kinase inhibitors include Sgk-derived peptide inhibitors such as dominant negative mutants of activated Sgk, antibody-derived inhibitors, known classes of serine/threonine kinase (e.g. PKC) inhibitors such as competitive inhibitors of ATP and substrate binding, antibiotics, etc., see Tables 1xe2x80x943. Activated Sgk specificity and activity are readily quantified, inter alia, in high throughput kinase assays.
In one embodiment, the inhibitor is a dominant negative mutant of activated Sgk. Such inhibitors are readily screened as described below. Exemplary dominant negative inhibitors are shown in Table 1.
In another embodiment, the inhibitor is an activated Sgk-specific antibody or antibody fragment, especially an intrabody (see below). Exemplary antibody-derived activated Sgk inhibitors are shown in Table 2.
In another embodiment, the invention provides small molecule inhibitors of activated Sgk. A wide variety of inhibitory compounds are readily identified in binding and binding inhibition assays as described below. Preferred inhibitors include natural compounds such as staurosporine (Omura S, et al. J Antibiot (Tokyo) July 1995;48(7):535-48), produced by a marine organism, and synthetic compounds such as PD 153035(4-(3-bromoanilino)-6,7-dimethoxyquinazoline), which also potently inhibits the EGF receptor protein kinase (Fry D W et al. Science Aug. 19, 1994;265(5175):1093-5). Members of the tyrphostin family of synthetic protein kinase inhibitors are also useful; these include compounds which are pure ATP competitors, compounds which are pure substrate competitors, and compounds which are mixed competitors: compete with both ATP and substrate (Levitzki A and Gazit A, Science Mar. 24, 1995;24267(5205):1782-8). Additional Sgk inhibitors include peptide-based substrate competitors endogenously made by the mammalian cell, e.g. PKI (protein kinase inhibitor, Seasholtz A F et al., Proc Natl Acad Sci USA Feb. 28, 1995;92(5):1734-8), or proteins inhibiting cdc kinases (Correa-Bordes J and Nurse P, Cell Dec. 15, 1995;83(6):1001-9). Additional small peptide based substrate competitive kinase inhibitors and allosteric inhibitors (inhibitory mechanisms independent of ATP or substrate competition) are readily generated by established methods (Hvalby O, et al. Proc Natl Acad Sci USA May 24, 1994;91(11):4761-5; Barja P, et al., Cell Immunol January 1994;153(1):28-38; Villar-Palasi C, Biochim Biophys Acta Dec. 30, 1994;1224(3):384-8; Liu W Z, et al., Biochemistry Aug. 23, 1994;33(33):10120-6). Exemplary small molecule activated Sgk inhibitors, identified in our screens are shown in Table 3.
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Accordingly, the invention provides methods for modulating signal transduction involving IxcexaB in a cell comprising the step of modulating Sgk kinase activity, e.g. by contacting the cell with a serine/threonine kinase inhibitor. The cell may reside in culture or in situ, i.e. within the natural host. Preferred inhibitors are orally active in mammalian hosts. For diagnostic uses, the inhibitors or other Sgk binding agents are frequently labeled, such as with fluorescent, radioactive, chemiluminescent, or other easily detectable molecules, either conjugated directly to the binding agent or conjugated to a probe specific for the binding agent.
In a particular embodiment, the contacting is effected by introducing into the cell a polynucleotide encoding the inhibitor under conditions whereby the inhibitor is expressed in the cell. A wide variety of widely known methods may be used for targeting, introducing and expressing the requisite polynucleotide.
In another embodiment, the invention provides methods of classifying a proliferating cell as subject to excess activated Sgk activity by detecting an above-normal amount of activated Sgk activity in the cell. For example, many undesirably proliferating transformed cells are readily shown to contain higher levels of activated Sgk and/or Sgk activity than their untransformed counterparts. A wide variety of methods may be used to detect the activated Sgk activity, including contacting the cell or a fraction of the cell with an activated Sgk-specific antibody or antibody fragment, as described below. In addition, these classification methods may be used in conjunction with the disclosed methods of inhibiting activated Sgk.
The invention also provides methods and compositions relating to agents which specifically interact with Sgk, including mixtures comprising isolated Sgk and an isolated Sgk-interacting protein (SIP), which may be identified, for example, in two-hybrid screens or coprecipitation studies, as described below. Exemplary SIPs include Pendulin, PS2 and IF-1. Novel Sgk binding agents are also readily screened from complex mixtures or libraries by conventional and/or high throughput screens. For example, the invention provides methods for identifying a modulator of binding of activated Sgk and an Sgk-interacting protein by (a) contacting a mixture of an Sgk and an SIP with a candidate agent under conditions wherein but for the presence of the agent, the Sgk specifically binds the Sgk-interacting protein at a reference affinity and (b) detecting the binding affinity of the Sgk to the Sgk-interacting protein to determine an agent-biased affinity, wherein a difference between the agent-biased affinity and the reference affinity indicates that said agent modulates the binding of Sgk with the Sgk-interacting protein.